The ferric Iron uptake transcriptional regulator (Fur) governs virulence and stress responses in Listeria monocytogenes and serves as a target for novel food safety interventions.

Listeria monocytogenes is a major foodborne pathogen whose environmental persistence and virulence pose significant threats to food safety. The ferric uptake regulator (Fur) is essential for iron homeostasis; however, its role in the acid and oxidative stress responses, pathogenicity and its potential as a target for food safety interventions remain poorly understand. In this study, a Δfur mutant was constructed to investigate these questions. Deletion of fur severely impaired survival under acidic stress and disrupted iron homeostasis, resulting in intracellular Fe2+ accumulation. The mutant also exhibited enhanced resistance to oxidative stress, elevated glutathione (GSH) levels, and increased swimming motility. Additionally, the mutant showed reduced adhesion, invasion, cell-to-cell spread, and phagosome escape which correlated with attenuated virulence and lower bacterial burdens in mice. Electrophoretic mobility shift assays (EMSA) and qRT-PCR analysis showed that Fur modulates the expression of some PrfA-regulon virulence genes by binding to the promoters of prfA and hly in the presence of Fe2+ and GSH. PrfA also bound to the fur promoter, suggesting a cross-regulatory loop between Fur and PrfA. In the wild-type strain, expression of fur and its downstream genes was upregulated under iron-limited conditions. Building on this regulatory insight, a Fur-overexpression strain CΔfur_Phelp was engineered. This strain displayed heightened sensitivity to stresses in PBS at both neutral (pH 7.4) and acidic (pH 3.5 or 5.5) conditions, and exhibited poor survival under refrigeration, particularly in milk. The iron chelator 2, 2'-dipyridyl (Dip) and sodium phytate inhibited bacterial viability in PBS at neutral pH, and the inhibitory effect of Dip was further enhanced under citric acid conditions (pH 3.5-5.5) upon prolonged incubation. This work establishes Fur as a master regulator linking metabolism to virulence, and proposes novel anti-listeria strategies targeting Fur or iron availability.